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Marc Vidal

Publications and source records attributed to Marc Vidal.

At least 37 records · Page 2Linked to original sources
PubMed2004

Evidence for dynamically organized modularity in the yeast protein-protein interaction network.

In apparently scale-free protein-protein interaction networks, or 'interactome' networks, most proteins interact with few partners, whereas a small but significant proportion of proteins, the 'hubs', interact with many partners. Both biological and non-biological scale-free networks are particularly resistant to random node removal but are extremely sensitive to the targeted removal of hubs. A link between the potential scale-free topology of interactome networks and genetic robustness seems to exist, because knockouts of yeast genes encoding hubs are approximately threefold more likely to confer lethality than those of non-hubs. Here we investigate how hubs might contribute to robustness and other cellular properties for protein-protein interactions dynamically regulated both in time and in space. We uncovered two types of hub: 'party' hubs, which interact with most of their partners simultaneously, and 'date' hubs, which bind their different partners at different times or locations. Both in silico studies of network connectivity and genetic interactions described in vivo support a model of organized modularity in which date hubs organize the proteome, connecting biological processes--or modules--to each other, whereas party hubs function inside modules.

Computer Simulation↗
PubMed2004

Systematic interactome mapping and genetic perturbation analysis of a C. elegans TGF-beta signaling network.

To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.

Animals↗
PubMed2004

BRCA1/BARD1 orthologs required for DNA repair in Caenorhabditis elegans.

Inherited germline mutations in the tumor suppressor gene BRCA1 predispose individuals to early onset breast and ovarian cancer. BRCA1 together with its structurally related partner BARD1 is required for homologous recombination and DNA double-strand break repair, but how they perform these functions remains elusive. As part of a comprehensive search for DNA repair genes in C. elegans, we identified a BARD1 ortholog. In protein interaction screens, Ce-BRD-1 was found to interact with components of the sumoylation pathway, the TACC domain protein TAC-1, and most importantly, a homolog of mammalian BRCA1. We show that animals depleted for either Ce-brc-1 or Ce-brd-1 display similar abnormalities, including a high incidence of males, elevated levels of p53-dependent germ cell death before and after irradiation, and impaired progeny survival and chromosome fragmentation after irradiation. Furthermore, depletion of ubc-9 and tac-1 leads to radiation sensitivity and a high incidence of males, respectively, potentially linking these genes to the C. elegans BRCA1 pathway. Our findings support a shared role for Ce-BRC-1 and Ce-BRD-1 in C. elegans DNA repair processes, and this role will permit studies of the BRCA1 pathway in an organism amenable to rapid genetic and biochemical analysis.

Amino Acid Sequence↗
PubMed2004

A map of the interactome network of the metazoan C. elegans.

To initiate studies on how protein-protein interaction (or "interactome") networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome (WI5) map contains approximately 5500 interactions. Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses.

Animals↗
PubMed2004

ORFeome projects: gateway between genomics and omics.

The availability of entire genome sequences is expected to revolutionize the way in which biology and medicine are conducted for years to come. However, achieving this promise still requires significant effort in the areas of gene annotation, cloning and expression of thousands of known and heretofore unknown protein-encoding genes. Traditional technologies of manipulating genes are too cumbersome and inefficient when one is dealing with more than a few genes at a time. Entire libraries composed of all protein-encoding open reading frames (ORFs) cloned in highly flexible vectors will be needed to take full advantage of the information found in any genome sequence. The creation of such ORFeome resources using novel technologies for cloning and expressing entire proteomes constitutes an effective gateway from whole genome sequencing efforts to downstream 'omics' applications.

Animals↗
PubMed2004

Increasing specificity in high-throughput yeast two-hybrid experiments.

Since its inception, the yeast two-hybrid (Y2H) system has proven to be an efficient system to identify novel protein-protein interactions. However, Y2H screens are sometimes criticized for generating high rates of false-positives. Minimizing false-positive interactions is especially important in proteome wide high-throughput (HT) Y2H. Here, we summarize various approaches that reduce false-positives in HT-Y2H projects. We evaluated the potential of examining putative positives after removing the prey encoding plasmid by negative selection. We found that this method reliably identifies false-positives caused by spontaneous conversion of baits into auto-activators and provides significant time-savings in HT screens. In addition, we present a method to eliminate an important source of false-positives: contaminating prey plasmids. Y2H interactors can be wrongly identified due to the presence of two or more different plasmids in the cells of a single yeast colony. Of these independent plasmids, only one encodes a genuine interactor. Contaminating plasmids are eliminated by extended culture of yeast cells under positive selection for the interaction, allowing the identification of the true interaction partner.

Animals↗
PubMed2004

The HUPO PSI's molecular interaction format--a community standard for the representation of protein interaction data.

A major goal of proteomics is the complete description of the protein interaction network underlying cell physiology. A large number of small scale and, more recently, large-scale experiments have contributed to expanding our understanding of the nature of the interaction network. However, the necessary data integration across experiments is currently hampered by the fragmentation of publicly available protein interaction data, which exists in different formats in databases, on authors' websites or sometimes only in print publications. Here, we propose a community standard data model for the representation and exchange of protein interaction data. This data model has been jointly developed by members of the Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organization (HUPO), and is supported by major protein interaction data providers, in particular the Biomolecular Interaction Network Database (BIND), Cellzome (Heidelberg, Germany), the Database of Interacting Proteins (DIP), Dana Farber Cancer Institute (Boston, MA, USA), the Human Protein Reference Database (HPRD), Hybrigenics (Paris, France), the European Bioinformatics Institute's (EMBL-EBI, Hinxton, UK) IntAct, the Molecular Interactions (MINT, Rome, Italy) database, the Protein-Protein Interaction Database (PPID, Edinburgh, UK) and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, EMBL, Heidelberg, Germany).

Database Management Systems↗
PubMed2004

Annotation transfer between genomes: protein-protein interologs and protein-DNA regulogs.

Proteins function mainly through interactions, especially with DNA and other proteins. While some large-scale interaction networks are now available for a number of model organisms, their experimental generation remains difficult. Consequently, interolog mapping--the transfer of interaction annotation from one organism to another using comparative genomics--is of significant value. Here we quantitatively assess the degree to which interologs can be reliably transferred between species as a function of the sequence similarity of the corresponding interacting proteins. Using interaction information from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Helicobacter pylori, we find that protein-protein interactions can be transferred when a pair of proteins has a joint sequence identity >80% or a joint E-value <10(-70). (These "joint" quantities are the geometric means of the identities or E-values for the two pairs of interacting proteins.) We generalize our interolog analysis to protein-DNA binding, finding such interactions are conserved at specific thresholds between 30% and 60% sequence identity depending on the protein family. Furthermore, we introduce the concept of a "regulog"--a conserved regulatory relationship between proteins across different species. We map interologs and regulogs from yeast to a number of genomes with limited experimental annotation (e.g., Arabidopsis thaliana) and make these available through an online database at http://interolog.gersteinlab.org. Specifically, we are able to transfer approximately 90,000 potential protein-protein interactions to the worm. We test a number of these in two-hybrid experiments and are able to verify 45 overlaps, which we show to be statistically significant.

Amino Acid Sequence↗
PubMed2004

A gateway-compatible yeast one-hybrid system.

Since the advent of microarrays, vast amounts of gene expression data have been generated. However, these microarray data fail to reveal the transcription regulatory mechanisms that underlie differential gene expression, because the identity of the responsible transcription factors (TFs) often cannot be directly inferred from such data sets. Regulatory TFs activate or repress transcription of their target genes by binding to cis-regulatory elements that are frequently located in a gene's promoter. To understand the mechanisms underlying differential gene expression, it is necessary to identify physical interactions between regulatory TFs and their target genes. We developed a Gateway-compatible yeast one-hybrid (Y1H) system that enables the rapid, large-scale identification of protein-DNA interactions using both small (i.e., DNA elements of interest) and large (i.e., gene promoters) DNA fragments. We used four well-characterized Caenorhabditis elegans promoters as DNA baits to test the functionality of this Y1H system. We could detect approximately 40% of previously reported TF-promoter interactions. By performing screens using two complementary libraries, we found novel potentially interacting TFs for each promoter. We recapitulated several of the Y1H-based protein-DNA interactions using luciferase reporter assays in mammalian cells. Taken together, the Gateway-compatible Y1H system will allow the high-throughput identification of protein-DNA interactions and may be a valuable tool to decipher transcription regulatory networks.

Animals↗
PubMed2004

Generation of the Brucella melitensis ORFeome version 1.1.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.

Bacterial Proteins↗
PubMed2004

Feasibility of genome-scale construction of promoter::reporter gene fusions for expression in Caenorhabditis elegans using a multisite gateway recombination system.

The understanding of gene function increasingly requires the characterization of DNA segments containing promoters and their associated regulatory sequences. We describe a novel approach for linking multiple DNA segments, here applied to the generation of promoter::reporter fusions. Promoters from Caenorhabditis elegans genes were cloned using the MultiSite Gateway cloning technology. The capacity for using this system for efficient construction of chimeric genes was explored by constructing promoter::reporter gene fusions with a gfp reporter. The promoters were found to provide appropriate expression of GFP upon introduction into C. elegans, demonstrating that the short Gateway recombination site between the promoter and the reporter did not interfere with transcription or translation. The recombinational cloning involved in the Gateway system, which permits the highly efficient and precise transfer of DNA segments between plasmid vectors, makes this technology ideal for genomics research programs.

Animals↗
PubMed2004

C. elegans ORFeome version 3.1: increasing the coverage of ORFeome resources with improved gene predictions.

The first version of the Caenorhabditis elegans ORFeome cloning project, based on release WS9 of Wormbase (August 1999), provided experimental verifications for approximately 55% of predicted protein-encoding open reading frames (ORFs). The remaining 45% of predicted ORFs could not be cloned, possibly as a result of mispredicted gene boundaries. Since the release of WS9, gene predictions have improved continuously. To test the accuracy of evolving predictions, we attempted to PCR-amplify from a highly representative worm cDNA library and Gateway-clone approximately 4200 ORFs missed earlier and for which new predictions are available in WS100 (May 2003). In this set we successfully cloned 63% of ORFs with supporting experimental data ("touched" ORFs), and 42% of ORFs with no supporting experimental evidence ("untouched" ORFs). Approximately 2000 full-length ORFs were cloned in-frame, 13% of which were corrected in their exon/intron structure relative to WS100 predictions. In total, approximately 12,500 C. elegans ORFs are now available as Gateway Entry clones for various reverse proteomics (ORFeome v3.1). This work illustrates why the cloning of a complete C. elegans ORFeome, and likely the ORFeomes of other multicellular organisms, needs to be an iterative process that requires multiple rounds of experimental validation together with gradually improving gene predictions.

Animals↗
PubMed2004

A first version of the Caenorhabditis elegans Promoterome.

An important aspect of the development of systems biology approaches in metazoans is the characterization of expression patterns of nearly all genes predicted from genome sequences. Such "localizome" maps should provide information on where (in what cells or tissues) and when (at what stage of development or under what conditions) genes are expressed. They should also indicate in what cellular compartments the corresponding proteins are localized. Caenorhabditis elegans is particularly suited for the development of a localizome map since all its 959 adult somatic cells can be visualized by microscopy, and its cell lineage has been completely described. Here we address one of the challenges of C. elegans localizome mapping projects: that of obtaining a genome-wide resource of C. elegans promoters needed to generate transgenic animals expressing localization markers such as the green fluorescent protein (GFP). To ensure high flexibility for future uses, we utilized the newly developed MultiSite Gateway system. We generated and validated "version 1.1" of the Promoterome: a resource of approximately 6000 C. elegans promoters. These promoters can be transferred easily into various Gateway Destination vectors to drive expression of markers such as GFP, alone (promoter::GFP constructs), or in fusion with protein-encoding open reading frames available in ORFeome resources (promoter::ORF::GFP).

Animals↗
PubMed2004

Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library.

The recently completed Caenorhabditis elegans genome sequence allows application of high-throughput (HT) approaches for phenotypic analyses using RNA interference (RNAi). As large phenotypic data sets become available, "phenoclustering" strategies can be used to begin understanding the complex molecular networks involved in development and other biological processes. The current HT-RNAi resources represent a great asset for phenotypic profiling but are limited by lack of flexibility. For instance, existing resources do not take advantage of the latest improvements in RNAi technology, such as inducible hairpin RNAi. Here we show that a C. elegans ORFeome resource, generated with the Gateway cloning system, can be used as a starting point to generate alternative HT-RNAi resources with enhanced flexibility. The versatility inherent to the Gateway system suggests that additional HT-RNAi libraries can now be readily generated to perform gene knockdowns under various conditions, increasing the possibilities for phenome mapping in C. elegans.

Animals↗
PubMed2004

High-throughput expression of C. elegans proteins.

Proteome-scale studies of protein three-dimensional structures should provide valuable information for both investigating basic biology and developing therapeutics. Critical for these endeavors is the expression of recombinant proteins. We selected Caenorhabditis elegans as our model organism in a structural proteomics initiative because of the high quality of its genome sequence and the availability of its ORFeome, protein-encoding open reading frames (ORFs), in a flexible recombinational cloning format. We developed a robotic pipeline for recombinant protein expression, applying the Gateway cloning/expression technology and utilizing a stepwise automation strategy on an integrated robotic platform. Using the pipeline, we have carried out heterologous protein expression experiments on 10,167 ORFs of C. elegans. With one expression vector and one Escherichia coli strain, protein expression was observed for 4854 ORFs, and 1536 were soluble. Bioinformatics analysis of the data indicates that protein hydrophobicity is a key determining factor for an ORF to yield a soluble expression product. This protein expression effort has investigated the largest number of genes in any organism to date. The pipeline described here is applicable to high-throughput expression of recombinant proteins for other species, both prokaryotic and eukaryotic, provided that ORFeome resources become available.

Animals↗
PubMed2004

ORFeome cloning and systems biology: standardized mass production of the parts from the parts-list.

Together with metabolites, proteins and RNAs form complex biological systems through highly intricate networks of physical and functional interactions. Large-scale studies aimed at a molecular understanding of the structure, function, and dynamics of proteins and RNAs in the context of cellular networks require novel approaches and technologies. This Special Issue of Genome Research features strategies for the high-throughput construction and manipulation of complete sets of protein-encoding open reading frames (ORFeome), gene promoters (promoterome), and noncoding RNAs, as predicted from genome and transcriptome sequences. Here we discuss the use of a recombinational cloning system that allows efficiency, adaptability, and compatibility in the generation of ORFeome, promoterome, and other resources.

Animals↗
PubMed2004

Human ORFeome version 1.1: a platform for reverse proteomics.

The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.

Cloning, Molecular↗